The non-tuberculous mycobacteria (NTM) are a growing public health concern as the number of opportunistic infections increases. Although not a reportable disease, there is growing epidemiologic evidence to suggest that NTM cause more infections today in the United States than Mycobacterium tuberculosis (Mtb) yet, unlike Mtb, there are few dedicated antimicrobial drug discovery programs specifically for NTM. Pulmonary disease caused by NTM is especially problematic in patients with underlying susceptibilities such as immunosuppressive medications, cystic fibrosis and other lung diseases, HIV and malignancies. Given the emergence of NTM as a public health issue, finding new antibacterial agents is of high importance. We have screened libraries of novel compounds for anti-NTM activity, focusing on M. abscessus, and have found several promising hits centered around three chemically tractable small molecule scaffolds that exhibit minimal cytotoxicity. The minimum inhibitory concentrations (MIC) values range from 0.5 to 4 g/mL for different NTM, thus providing a relatively potent starting point for optimization. Importantly, each of these compounds also has activity against Mtb, enabling us to focus on broad-spectrum activity. We propose to perform medicinal chemistry hit-to-lead optimization, developing an understanding of the key structure-activity relationships driving antibacterial activity. In Aim 1, we will synthesie a small set of analogs for each of three scaffolds, focusing on efficient sites for derivatization tht will allow for rapid synthesis of 30-50 analogs. In addition, there are 76 commercially available analogs within these compound series, facilitating rapid assembly of at least 100 analogs for testing in Aim 2. The goal of Aim 2 is to prioritize the scaffolds based on biological properties, beginning with MIC testing against a panel of NTM, including M. abscessus, M. avium, M. intracellulare, and M. chelonae. Analogs that show anti-NTM activity will progress to a secondary screen against Mtb and a broad panel of clinically important Gram-positive and Gram-negative pathogens and fungi. Further characterization will include analysis of metabolic stability in human liver microsomes, assessment of bactericidal potential, serum protein binding potential and sensitivity to efflux inhibitors. The most promising scaffold with respect to breadth of antimicrobial spectrum, potency and pharmacologic properties will be selected for in-depth medicinal chemistry optimization in Aim 3, utilizing an iterative cycle of chemical synthesis followed by biological profiling to guide the development of SAR. Successful completion of Aim 3 is expected to produce one or more compound leads suitable for future in vivo testing. We believe these compounds represent an exciting and promising starting point for the development of a novel therapeutic agent with broad antimycobacterial activity.